Most bacterial viruses ("bacteriophages" or just "phages") are highly efficient in their ability to infect their often very specific bacterial hosts. In general, only one or a few viral particles are necessary to successfully infect one bacterium. As a consequence there has been a long, unfulfilled hope that phages could be used as antibiotics. The need for alternative antibiotics is becoming ever more acute as common bacterial pathogens are developing resistant mutations, making it urgent to develop alternatives such as phage therapy. In addition, recombinant phages have been suggested as a means for gene therapy and as potential antigens for development of vaccines. Bacteriophage T4 has been a model system to study virus structure and function, having advanced biochemical principles of the assembly of biological complexes and protein-protein interactions. We have extensive experience in both structural and functional studies of phage T4. We now wish to expand this knowledge and make it available for potential medical applications. The plan is to analyze the structure and assembly of the capsid (Specific Aim 1), the DNA packaging machine (Specific Aim 2), the tail assembly (Specific Aim 3), assembly of the tail with head (Specific Aim 4) and the recognition of the host by the phage fibers (Specific Aim 5). Our primary tools will be molecular biology, protein chemistry, crystallography and electron microscopy. Molecular biology studies will produce pure samples in sufficient quantity for structural and functional studies. Crystallographic studies will be of protein components, providing three-dimensional information at near atomic resolution. Cryo-electron microscopic reconstructions will provide three-dimensional data on the organization of the protein components within the virus. Combining these techniques will generate "pseudo-atomic" resolution structures of the virus at different stages of its life cycle. PUBLIC HEALTH RELEVANCE: We plan to extend the structural and functional knowledge of bacteriophage T4, which would advance basic knowledge of protein-protein interactions and macromolecular assembly. At this stage we do not directly plan work on medical applications, yet T4 is perhaps the most thoroughly studied phage and, thus, is the most likely candidate for developing its potential as an antibiotic, as a vaccine, and for the delivery of genes to targeted cells for gene therapy. The research will center on the structure of the phage tail and fibers relevant for host recognition, the mechanism of the DNA packaging motor relevant for gene delivery and therapy, and the structure of the capsid relevant for vaccine development.